In utero ethanol exposure causes long-term neurobehavioral dysfunction. The cognitive disabilities observed suggest that ethanol may interfere with synapse formation in the hippocampus of the developing brain. Several studies have shown that prenatal ethanol exposure causes a decrease in synaptogenesis; however, recent data suggests the opposite. Interestingly, none of the studies investigate the role of astrocytes in mediating the ethanol effect on hippocampal neuron synapse formation, although the role of astrocytes in the process is now well recognized. This is the general aim of this proposal, making it a novel and important investigation. Using an in vitro co-culture system, primary hippocampal neurons (14 DIC) are incubated for 24 hours with astrocytes pre-treated with ethanol (75 mM). To quantify the effect on synapse formation, neurons are labeled for the pre- and post-synaptic proteins synaptophysin and PSD-95. Three-dimensional object analysis is used to analyze the number of pre- and post-synaptic puncta and their overlap. Preliminary work has shown that ethanol treatment of astrocytes significantly increases the number of both pre- and post-synaptic puncta, and causes a slightly higher than 4-fold increase in their overlap, suggesting an effect on synapse formation. Additionally, western blot analysis has shown that ethanol pre-treatment of astrocytes significantly increased levels of both synaptic proteins relative to control. These data suggest that ethanol-treated astroctyes may release factors that enhance synaptic formation. Studies using the methods described above, as well as new techniques are proposed to characterize this observed effect. Aim 1 of this proposal will expand on the preliminary findings and characterize the effect of ethanol-treated astrocytes on synaptogenesis in hippocampal neurons. Experiments are planned to study the effects of lower doses of ethanol, a time course to determine if the effects are long-lasting, assessment of mRNA transcript levels, and electrophysiology studies to determine if the observed synapses are functional. Aim 2 will test the hypothesis that increased cholesterol release from ethanol-treated astrocytes is responsible for the observed ethanol- modulation of synaptic structure formation. To show that cholesterol is sufficient to cause the observed effect, cholesterol-containing lipoprotein particles will be added to the neuronal cultures, and cholesterol release from astrocytes will be pharmacologically increased in the co-culture system. Additionally, cholesterol will be shown to be a necessary component in producing the effect through experiments where astrocyte cholesterol synthesis will be blocked in the co-culture system and those that block the uptake of cholesterol- containing lipoproteins in neurons. Together, the findings of this proposal will increase understanding of the astrocyte-mediated mechanisms involved in the altered neuronal development caused by ethanol. These findings may suggest potential treatments for those children affected by in utero ethanol exposure. PUBLIC HEALTH RELEVANCE: This proposal will investigate the role of astrocytes in mediating the effect of ethanol on the formation of synapses in the developing brain. This study has implications for the cognitive dysfunction found in children with Fetal Alcohol Spectrum Disorder (FASD). Increased understanding in this area could lead to potential treatments for children exposed in utero to ethanol.